Human serine proteases and serine proteases from mammals are involved in numerous physiological processes (Barrett, A. J., Methods in Enzymology, Vol. 244 (1994) Academic Press, New York; Twining, S. S., Crit. Revs. Biochem. Mol. Biol. 29 (1994) 315-383). These are essentially protein digestion, blood coagulation (Davie, E. W. et al., Biochemistry 20 (1991) 10363-10370), fertilization (Baba, T., FEBS Letters 27 (1989) 296-300), programmed cell death as well as complement activation in the immune response (Goldberger, G. et al., J. Biol. Chem. 262 (1987) 10065-10071). Furthermore serine proteases are known from insect cells (Gay, N. J. et al., Biochim. Biophys. Acta 1132 (1992) 290-296), from viruses (Allaire, M. et al., Nature 369 (1994) 72-76) as well as from prokaryotes. Prokaryotic serine proteases are for example subtilisin (Kraut, J., in The Enzymes (Boyer, P. D., ed.) Vol. 3, 547-560 (1971) Academic Press, New York and London), carboxypeptidase II (Liao, D. et al., Biochemistry 31 (1992) 9796-9812) and Streptomyces griseus trypsin (Read, R. J. and James, M. N. G., J. Mol. Biol. 200 (1988) 523-551).
Blood homoeostasis, the equilibrium between blood coagulation and fibrinolysis is ensured by several very complex systems which mutually influence each other. In this connection proteases play a role in blood coagulation, closure of wounds by fibrin formation as well as in fibrinolysis, i.e. clot lysis. After an injury the xe2x80x9cinjury signalxe2x80x9d is amplified by sequential activation (specific proteolysis) of inactive proenzymes to active enzymes which initiates blood coagulation and ensures a rapid closure of wounds. Blood coagulation (haemostasis) can be initiated by two paths, the intrinsic path in which all protein components are present in the blood, and the extrinsic path in which a membrane protein, the so-called tissue factor, plays a critical role. The molecular mechanism of blood homoeostasis and the components that are involved in this has been comprehensively described in several review articles (Furie, B. et al., Cell 53 (1988) 505-518; Davie, E. W. et al., Biochem. 30 (1991) 10363-10379; Bergmeyer, H. U. (ed.): Methods of Enzymatic Analysis, Vol. V, chapter 3, 3rd ed., Academic Press, New York (1983)).
If the blood homoeostasis becomes unbalanced (blood coagulation versus fibrinolysis), an increased coagulation tendency of the blood can lead to various thrombotic disorders/diseases such as e.g. deep-vein thrombosis, pulmonary embolism, cardiac infarction and stroke (Mustard, J. F. et al., In: Haembstasis and Thrombosis. Bloom, A. L. and Thomas, D. P. (eds), 2nd edition, Churchill-Livingstone, Edinburgh, (1987) pp. 618-650). Coagulation disorders with bleeding such as e.g. in haemophilia A (defective factor VIII) and haemophilia B (defective factor IX) can occur as a result of a reduced tendency of the blood to coagulate.
There is therefore a need for substances which can influence the system of blood coagulation and fibrinolysis according to the medical needs. Factor VIII or factor IX or recently also factor VII isolated from the blood or produced recombinantly is used for example to treat haemophilia A and B. tPA (tissue type plasminogen activator) and streptokinase (bacterial plasminogen activator) are used to lyse clots for example after cardiac infarction. Antithrombotic substances (Harker, L. A. et al., In: Hemostasis and Thrombosis: Basic Principles and Clinical Praxis, Colman, R. W. et al., (eds.) 3rd edition, Lippincott, Philadelphia, (1994) pp. 1638-1660) such as e.g. hirudin (peptide composed of 65 amino acids, specific thrombin inhibitor; Maraganore, J. M., Thrombosis and Haemostasis 70 (1993) 208-211), heparin (heteroglycan, cofactor of endogenous inhibitors; Barrowcliffe, T. W. et al., In: Haemostasis and Thrombosis. Bloom, A. L. et al. (eds.); 3rd edition, Churchill-Livingstone, Edinburgh (1994) Vol. 2, pp. 1417-1438) and oral vitamin K antagonists (inhibitors of xcex3-carboxylation; Glu residues of the Gla domain; Hirsh, J. et al., In: Hemostasis and Thrombosis, Basic Principles and Clinical Praxis, Colman, R. W. et al., (eds.), 3rd edition, Lippincott, Philadelphia, (1994) pp. 1567-1583) are used to inhibit blood coagulation. However, the available substances are often still very expensive (protein factors) and/or not ideal with regard to their medical application and lead to considerable side effects.
All antithrombotic substances interfere with one or usually even several targets within the blood coagulation cascade. The inevitable price paid for a partial inactivation of the haemostatic system by antithrombotic substances is an increased risk of bleeding. The orally available vitamin K antagonists interfere with all vitamin K dependent coagulation factors such as e.g. the blood plasma proteases FVII, FIX, FX and thrombin which have a Gla domain that is post-translationally modified by xcex3-carboxylation. Consequently this antithrombotic therapy is very unspecific and influences the intrinsic as well as the extrinsic haemostatic system. Like the vitamin K antagonists, heparin interferes with several targets within the blood coagulation cascade. The antithrombotic action is due to an increased inactivation of for example thrombin, FIXa and FXa by an increased rate of formation of the complex with the natural inhibitor antithrombin III. Even the specific thrombin inhibitor hirudin derived from the leech has failed in clinical studies due to frequently occurring bleeding. There is therefore a need for new selective and better tolerated antithrombotic substances with an improved benefit to side effect ratio. In this connection the inhibition of the FXa mediated activation of prothrombin to thrombin by specific FXa inhibitors appears to be an attractive target.
The search for new modulators (activators, inhibitors) of blood coagulation, fibrinolysis and homoeostasis can be carried out by screening libraries of substances and optionally subsequently improving an identified lead structure by drug modelling. For this it is necessary that the serine proteases according to the invention are available in a crystalline form.
Attractive targets within blood homoeostasis are for example the activated serine proteases thrombin, FVIIa, FIXa, FXa, FXIa; FXIIa; kallikrein (blood coagulation), tPA, urokinase, plasmin (fibrinolysis) and activated protein C (regulatory anticoagulant) and inactive precursors (zymogens) thereof. Furthermore the complexes which form by interaction between a blood plasma protease and cofactor during blood homoeostasis such as for example FXa::FVa, FIXa::FVIIIa, thrombin::thrombomodulin, FVII/FVIIa::tissue factor are also of interest as a target.
Serine proteases can be produced recombinantly by biotechnological methods. Examples of this are human tissue plasminogen activator, urokinase and subtilisin. However, it has turned out that the serine proteases isolated from natural sources as well as those produced recombinantly do not fulfil all requirements with regard to substrate specificity, stability and purity that are needed for therapeutic applications or when they are used to cleave fusion proteins in biotechnological production processes. In particular the serine proteases factor Xa and kexin (kex 2) are very unstable. Proteases isolated from animal and/or human raw materials such as e.g. trypsin, thrombin, factor IXa and factor Xa are problematic for a therapeutic application or for an application in a production process for therapeutics since they may be contaminated with human pathogenic agents such as e.g. viruses and/or prions.
Moreover proteases isolated from animal and/or microbial raw materials are very often additionally contaminated with undesired host cell proteases. For this reason the trypsin from animal raw materials that is used to process insulin is treated with L-1-tosylamide-2-phenyl-ethyl-chloromethyl ketone (TPCK) (Kemmler, W. et al., J. Biol. Chem. 246 (1971) 6786-6791) in order to inhibit the chymotrypsin activity in these preparations. Factor Xa preparations are usually contaminated with thrombin. In the purification of lysyl endoproteinase from lysobacter, the xcex1-lytic protease has to be separated by very complicated process steps.
A chimeric protein comprising a first sequence and a second sequence C-terminal to the first sequence and linked to the first sequence by one or more peptide bonds, the first sequence having the amino acid sequence of the first catalytic domain half of a first serine protease and the second sequence having the amino acid sequence of the second catalytic domain half of a second serine protease different from the first serine protease.